Holographic Dark Energy Models and Higher Order Generalizations in Dynamical Chern-Simons Modified Gravity

Dark Energy models are here investigated and studied in the framework of the Chern-Simons modified gravity model. We bring into focus the Holographic Dark Energy (HDE) model with Granda-Oliveros cut-off, the Modified Holographic Ricci Dark Energy (MHRDE) model and, moreover, a model with higher derivatives of the Hubble parameter as well. The relevant expressions of the scale factor a(t) for a Friedmann-Robertson-Walker Universe are derived and studied, and in this context, the evolution of the scale factor is shown to be similar to that one displayed by the modified Chaplygin gas in two of the above models.Comment: 7 pages, to appear in Eur. Phys. J.

Improving AAV manufacturing by integrated and continuous processing

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Insect cell-based production processes intensified via high cell density perfusion and continuous culture

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Insect cell platforms for production of pseudo-typed VLPs for drug and vaccine development

Conformational-complex membrane proteins (MPs) are vaccine/drug targets in many diseases, but drug and vaccine development has been slowed down by the lack of efficient production tools. Co-expression of MPs with matrix proteins from enveloped viruses is a promising approach to obtain correctly folded proteins at the surface of ordered nanoscale architectures such as virus-like particles (VLPs), preserving their native lipidic environment. In this work, we implemented an innovative site-specific recombination strategy based on flipase-mediated cassette exchange technology to establish reusable insect cell platforms for fast production of enveloped VLPs pseudo-typed with target MPs. Influenza M1 and HIV Gag proteins were evaluated as scaffolds, and proof-of-concept (PoC) demonstrated using two membrane proteins, the influenza HA protein (e.g. for vaccines) and the human beta-2 adrenergic receptor (e.g. for drug screening or antibody discovery). Bioprocess engineering schemes were designed (adaptive laboratory evolution to hypothermic culture conditions and supplementation with productivity enhancers), allowing to improve HIV Gag-VLPs production in the developed stable insect cells. Under hypothermic culture conditions, adapted cells expressed up to 30-fold more HIV Gag-VLPs than non-adapted cells. Noteworthy, the element driving such increase in productivity is the adaptation process and not the temperature shift as the later alone leads to lower production yields. A more modest increase in productivity (up to 7-fold) was observed when supplementing non-adapted cell cultures with productivity enhancers NaBu and DMSO. PoC was successfully demonstrated in 0.5 L stirred-tank bioreactors. Profiting from the platforms developed above, a modular system comprising stable and baculovirus-mediated expression in insect cells was established for the production of a multi-HA influenza VLP as vaccine candidate that otherwise could not be obtained due to baculovirus vector instability. By combining stable with transient expression systems, we could rationally distribute the number of genes to be expressed per platform and thus generate the target VLP for subsequent animal studies. In addition, a tailor-made refeed strategy was designed based on the exhaustion of key nutrients during cell growth resulting in a 4-fold increase in HA titers per mL. PoC was successfully demonstrated in 2 L stirred-tank bioreactors. Overall, the insect cell platforms and bioprocess engineering strategies herein assembled have the potential to assist/accelerate drug and vaccine development. Acknowledgments: This work was supported by European Commission (Project EDUFLUVAC, Grant nr. 602640) and by Portuguese “Fundação para a Ciência e a Tecnologia” through the following programs: FCT Investigator Starting Grant (IF/01704/2014), Exploratory Research and Development Project EXPL/BBB-BIO/1541/2013, and PhD fellowships SFRH/BD/86744/2012 and SFRH/BD/90564/2012

Bioprocess engineering of insect cells for accelerating vaccines development

Insect cells emerged as a powerful and versatile platform for vaccines production, mostly using the lytic baculovirus expression vector system (BEVS). Stable expression in such hosts has been increasingly explored to circumvent BEVS-related drawbacks, but protein titers achieved to date are still seemingly low. The design of new or improved cell factories and bioprocess intensification strategies are therefore necessary to increase productivities and thus accelerate implementation of stable insect cell lines as a fast, cost-effective platform for vaccines manufacturing. In this work, we implemented an innovative site-specific recombination strategy based on flipase-mediated cassette exchange technology to establish reusable insect (Sf-9 and High Five) cell platforms for fast production of enveloped virus-like particles (VLPs). Influenza M1 and HIV Gag proteins were evaluated as scaffolds, and proof-of-concept demonstrated using two membrane proteins: the influenza HA protein (for vaccines) and the human beta-2 adrenergic receptor (for drug screening or antibody discovery). Aiming to improve production yields in developed stable cell lines, two bioprocess engineering schemes were evaluated (either individually or in combination): (i) adaptive laboratory evolution of insect cells to hypothermic culture conditions, and (ii) supplementation of insect cell cultures with productivity enhancers. The stable cell line expressing HIV Gag-VLPs was used as model. Under hypothermic culture conditions, adapted Sf-9 cells expressed up to 30-fold more HIV Gag-VLPs than non-adapted cells. Noteworthy, the element driving such increase in productivity is the adaptation process and not the temperature shift as the latter alone leads to lower production yields. A more modest increase in productivity (up to 7-fold) was observed when supplementing non-adapted cell cultures with productivity enhancers NaBu and DMSO. No synergistic effect was observed when combining adapted cells and supplementation with productivity enhancers. Production of HIV Gag-VLPs was successfully scaled-up to stirred-tank bioreactors. The adapted cell line was then pseudo-typed with influenza HA protein for production of Gag-HA VLPs, and their performance benchmarked against (i) parental Sf-9 cells stably expressing Gag-HA VLPs and (ii) insect cells-BEVS, both cultured under standard temperature conditions (27C). Adapted cells showed increased production of Gag-HA VLPs when compared to parental/stable cells, corroborating previously obtained data, but still lower when compared to insect cells-BEVS. Bioprocess intensification strategies are currently under in-house testing to further improve yields of adapted cells and thus shorten the gap between stable insect cells and IC-BEVS. Overall, the insect cell platforms and bioprocess engineering strategies herein assembled have the potential to assist and accelerate vaccines development. Acknowledgments: This work was supported by European Commission (Project EDUFLUVAC, Grant nr. 602640) and by Portuguese “Fundação para a Ciência e a Tecnologia” through the following programs: FCT Investigator Starting Grant (IF/01704/2014), Exploratory Research and Development Project EXPL/BBB-BIO/1541/2013, and PhD fellowships SFRH/BD/86744/2012 and SFRH/BD/90564/2012

Intensification of viral vector production and clarification by integration of perfusion platforms

Gene Therapies represent an important new frontier in therapeutic development as they have the potential to treat diseases that have previously been difficult to manage. The new gene therapies currently being developed will address larger patient population and/or using higher dosage needed for global clinical and commercialization. Manufacturing improvement is therefore needed to realize the full potential of gene therapy. We will present two case studies which illustrate the next generation viral vector manufacturing process. The first case study will demonstrate how integration of perfusion platforms, alternating tangential flow (ATF) and the tangential flow depth filtration (TFDF) to bioreactor led to 2-4-fold increase of total rAAV8 yield comparing to the current bioreactor process. The second case study will show how the perfusion TFDF platform used in clarification mode was applied to increase lentivirus (LV) yield through multiple harvests. The cell retention filters enabled the continuous harvest clarification of LV particles present in the media during the virus production, demonstrating the potential for continuous upstream-downstream processing of secreted LV vectors. The implementation of the TFDF in a continuous clarification strategy during the harvest led to a total yield of more than 200% potent LV compared to the depth filtration process. Please click Additional Files below to see the full abstract

Analysis of the Impact of Size of Brain Metastases in the Overall Survival of Patients with Primary Head and Neck Cancer, Melanoma and Sarcoma

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Accelerating iterative methods for solving systems of linear equations using FPGAs

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